A reassessment of the effect of activated Notch1 on CD4 and CD8 T cell development

The Notch signaling pathway plays an important role in the early steps of T cell development and in the generation of T cell tumors, but its role in the CD4 vs CD8 lineage decision is controversial. Notch1 is not essential for CD4 or CD8 T cell development; however, there are suggestions that multiple Notch family members may act in a redundant fashion during thymic development. In theory, expressing a constitutively activated form of Notch in CD4(+)CD8(+) thymocytes could provide clues about the normal role of Notch in developing CD4 and CD8 T cells. Unfortunately, two different studies of transgenic mice expressing activated forms of Notch1 (Notch1IC) led to conflicting conclusions. In this study, we re-examine the effect of the two Notch1IC transgenes on thymocyte development. We find that both Notch1IC transgenic lines display a decrease in CD4 single positive (SP) thymocytes and a corresponding increase in CD8 SP thymocytes. The enhanced development of CD8 SP thymocytes is dependent on either class I or II MHC. Thus, data from two different Notch1IC transgenic lines indicate that Notch activity promotes CD8 and inhibits CD4 SP development. We suggest that the discrepancies in previous reports of Notch1IC transgenic mice are due to differences in the propensity of the two different transgenic lines to develop tumors.     

Myxoma virus induces extensive CD4 downregulation and dissociation of p56lck in infected rabbit CD4+ T lymphocytes.

Myxoma virus is a pathogenic poxvirus that induces extensive dysregulation of cellular immunity in infected European rabbits. Infection of a rabbit CD4+ T-cell line (RL-5) with myxoma virus results in dramatic reductions of cell surface levels of CD4 as monitored by flow cytometry. The virus-induced downregulation of CD4 requires early but not late viral gene expression and could not be inhibited by staurosporine, an inhibitor of protein kinase C, which effectively blocks phorbol 12-myristate-13-acetate-induced downregulation of CD4. The decrease in total cellular levels of CD4 during myxoma virus infection could be inhibited by the lysosomotrophic agent NH4Cl, suggesting a lysosomal fate for CD4 during myxoma virus infection. Steady-state levels of the CD4-associated protein tyrosine kinase p56lck remained unchanged during myxoma virus infection, suggesting that p56lck dissociates from CD4 prior to CD4 degradation in virus infected cells. Total p56lck kinase activity was unaffected during myxoma virus infection, although the amount of p56lck physically associated with CD4 declined in parallel with the loss of CD4. Thus, myxoma virus infection of CD4+ T lymphocytes triggers CD4 downregulation via a protein kinase C-independent pathway, causing the dissociation of p56lck and the degradation of CD4 in lysosomal vesicles.     

Interleukin-dependent modulation of HLA-DR expression on CD4and CD8 activated T cells

Summary Interleukins (IL) regulate different T-cell surface Ag known as activation markers that have distinct functional roles. In this paper, while studying the influence of some cytokines(IL-12, IL-2 and IL-4) on the expression of several markers [CD69,CD25, CD26, CD3, human leukocyte antigen (HLA-DR), CD45R0] in in vitro activated human T lymphocytes, we observed two groups of donors responding to phytohaemagglutinin (PHA) activation with high or low HLA-DRAg expression. We also found that CD4 and CD8 populations had different HLA-DR densities under PHA activation (particularly the high HLA-DR-expressing group). Interleukins, in a dose-dependent manner (IL-2 partially),upregulated these HLA-DR levels. In 5 day cultures, IL-12 and IL-2 enhanced the CD8/CD4 ratio of activated T cells,which was responsible, in part, for the IL-dependent HLA-DR upregulation.IL-12 and IL-2 also upregulated the HLA-DR expression at the molecular level on CD8, and IL-12 downregulated it on CD4 cells. It seems that IL-4 upregulated HLA-DR by shortening the mitogen-dependent regulation kinetics. We hypothesize that the different effect of each IL on HLA-DR expression might be related to the regulation of the dose of antigenic peptide presentation and, thus, also influence TH1/TH2 dominance.     

Biochemical differences in the alphabeta T cell receptor.CD3 surface complex between CD8+ and CD4+ human mature T lymphocytes

We have reported the existence of biochemical and conformational differences in the alphabeta T cell receptor (TCR) complex between CD4(+) and CD8(+) CD3gamma-deficient (gamma(-)) mature T cells. In the present study, we have furthered our understanding and extended the observations to primary T lymphocytes from normal (gamma(+)) individuals. Surface TCR.CD3 components from CD4(+) gamma(-) T cells, other than CD3gamma, were detectable and similar in size to CD4(+) gamma(+) controls. Their native TCR.CD3 complex was also similar to CD4(+) gamma(+) controls, except for an alphabeta(deltaepsilon)(2)zeta(2) instead of an alphabetagammaepsilondeltaepsilonzeta(2) stoichiometry. In contrast, the surface TCRalpha, TCRbeta, and CD3delta chains of CD8(+) gamma(-) T cells did not possess their usual sizes. Using confocal immunofluorescence, TCRalpha was hardly detectable in CD8(+) gamma(-) T cells. Blue native gels (BN-PAGE) demonstrated the existence of a heterogeneous population of TCR.CD3 in these cells. Using primary peripheral blood T lymphocytes from normal (gamma(+)) donors, we performed a broad epitopic scan. In contrast to all other TCR.CD3-specific monoclonal antibodies, RW2-8C8 stained CD8(+) better than it did CD4(+) T cells, and the difference was dependent on glycosylation of the TCR.CD3 complex but independent of T cell activation or differentiation. RW2-8C8 staining of CD8(+) T cells was shown to be more dependent on lipid raft integrity than that of CD4(+) T cells. Finally, immunoprecipitation studies on purified primary CD4(+) and CD8(+) T cells revealed the existence of TCR glycosylation differences between the two. Collectively, these results are consistent with the existence of conformational or topological lineage-specific differences in the TCR.CD3 from CD4(+) and CD8(+) wild type T cells. The differences may be relevant for cis interactions during antigen recognition and signal transduction.     

Production of lymphokine mRNA by CD45R+ and CD45R- helper T cells from human peripheral blood and by human CD4+ T cell clones

The expression of lymphokine mRNA by human CD4+CD45R+ and CD4+CD45R- Th cells was assessed after mitogen stimulation. These Ag have previously been shown to relate closely to virgin and primed T cells, respectively. CD4+CD45R+ (virgin) and CD4+CD45R- (primed) cell fractions were isolated by sorting double-labeled cells with a fluorescence-activated cell sorter. CD4+CD45R+ cells produced high levels of IL-2 mRNA when stimulated with either PMA together with calcium ionophore, or with PHA, but they expressed only trace quantities of mRNA for IL-4 or IFN-gamma. In contrast, CD4+CD45R- cells produced high levels of mRNA for IL-2, IL-4, and IFN-gamma. After 14 days of continuous culture, CD4+CD45R+ Th cells lost expression of the CD45R Ag, but gained high level expression of CDw29, such that they were indistinguishable from the cell population which originally expressed this Ag. At the same time, they acquired the ability to synthesize IL-4 mRNA. It seemed likely that the broad lymphokine profile of primed Th cells might mask clonal heterogeneity. Analysis of 122 CD4+ T cell clones showed that all of them synthesized IL-2 mRNA. One clone failed to express IL-4 mRNA, but did produce those for IL-2 and IFN-gamma. A total of 34 of the clones was investigated to determine expression of IFN-gamma mRNA; two of these clones were negative for IFN-gamma mRNA, and both expressed IL-2 and IL-4 message. These data suggest that while fresh virgin and primed peripheral blood T cells show a clear resolution of lymphokine production, a simple subdivision of human CD4+ T cell clones on the basis of their lymphokine production (such as that reported for mouse Th cell clones) is not possible.     

Function of the CD4 and CD8 molecules on human cytotoxic T lymphocytes: regulation of T cell triggering

The function of the T cell differentiation antigens CD4 (Leu-3/T4) and CD8 (Leu-2/T8) on human cytotoxic T lymphocytes (CTL) is presently seen only in conjugate formation between CTL and target cell via class II or class I MHC antigens rather than in the later killing steps. In this study, human CD4+ and CD8+ CTL clones were used to investigate the effects of monoclonal antibodies against these differentiation antigens on nonspecific triggering of cytotoxicity. Cytotoxicity was induced either by antibodies against the CD3 (T3) antigen or by the lectins Con A and PHA. Anti-CD4 or anti-CD8 antibodies specifically inhibited all types of cytotoxicity of CD4+ or CD8+ CTL, respectively, regardless of the specificity of the CTL for class I or class II HLA antigens and regardless of whether target cells expressed class I or class II antigens. These results are incompatible with an exclusive role of the CD4 and CD8 molecules in MHC class recognition and are discussed with respect to a function as negative signal receptors for these molecules on CTL.     

Simultaneous production of IL-2, IL-4, and IFN-gamma by activated human CD4+ and CD8+ T cell clones

In the present study, we have investigated the ability of human T cells to secrete IL-2, IL-4, and IFN-gamma. IL-4 and IFN-gamma were quantified with enzymatic immunoassays and IL-2 with a biologic assay by using the murine IL-2-dependent cell line CTLL-2. PBL, stimulated with Con A or with a combination of the phorbol ester 13-O-tetradecanoylphorbol-12-acetate and the Ca2+ ionophore A23187 secreted IL-2, IL-4, and IFN-gamma. The kinetics of the secretion of the three lymphokines was investigated with two CD4+ clones; one (GEO-2) that produced IL-2, IL-4, and IFN-gamma and another (HY640), that produced only IL-2 and IFN-gamma. Significant IL-2, IL-4, and IFN-gamma production was observed after only 8 h of activation. Maximal levels of IL-2 and IL-4 were found 20 h after the onset of the stimulation which subsequently decreased. In contrast, IFN-gamma levels continued to increase in a period up to 40 h and then leveled off. In spite of these differences in secretion, the kinetics of accumulation of mRNA did not differ. The IL-2, IL-4, and IFN-gamma mRNA were detectable 2 h after stimulation and continued to accumulate for a period up to 20 h. In a series of 22 CD4+ clones, 21 were able to secrete all three lymphokines upon stimulation. Almost all CD8+ clones were able to produce IL-2 and IFN-gamma, but only six of the 23 CD8+ T cell clones secreted IL-4. In addition, five CD4+ (allo)antigen-specific T cell clones were tested for IL-2, IL-4, and IFN-gamma secretion upon specific stimulation. Two alloantigen-specific and two tetanus toxoid-specific T cell clones secreted IL-2, IL-4, and IFN-gamma simultaneously, whereas one alloantigen-specific T cell clone secreted IL-2 and IFN-gamma, but not IL-4. A supernatant of the CD4+ T cell clone GEO-2, that contained high levels of IFN-gamma and IL-4, was unable to induce the low affinity receptor for IgE, CD23, on a Burkitt lymphoma cell line. However, after separation of IL-4 from IFN-gamma by using HPLC, the IL-4-containing fraction-induced CD23, which could be blocked by the fraction that contained IFN-gamma and by a polyclonal rabbit anti-IL-4 antiserum. Finally, the partly purified IL-4, that was devoid of IL-2, promoted the growth of the clone GEO-2.     

P-, E-, and L-selectin mediate migration of activated CD8+ T lymphocytes into inflamed skin

P- and E-selectin mediate CD4+ Th1 cell migration into the inflamed skin in a murine contact hypersensitivity model. In this model, not only CD4+ T cells but also CD8+ T cells infiltrate the inflamed skin, and the role of CD8+ type 1 cytotoxic T (Tc1) cells as effector cells has been demonstrated. Here we show that in mice deficient in both P- and E-selectin, the infiltration of CD8+ T cells in the inflamed skin is reduced, suggesting the role of these selectins in CD8+ T cell migration. We directly studied the role of selectins using in vitro-generated Tc1 cells. These cells are able to migrate into the inflamed skin of wild-type mice. This migration is partially mediated by P- and E-selectin, as shown by the reduced Tc1 cell migration into the inflamed skin of mice deficient in both P- and E-selectin or wild-type mice treated with the combination of anti-P-selectin and anti-E-selectin Abs. During P- and E-selectin-mediated migration of Tc1 cells, P-selectin glycoprotein ligand-1 appears to be the sole ligand for P-selectin and one of the ligands for E-selectin. P- and E-selectin-independent migration of Tc1 cells into the inflamed skin was predominantly mediated by L-selectin. These observations indicate that all three selectins can mediate Tc1 cell migration into the inflamed skin.     

Valpha24-JalphaQ-independent,CD1d-restricted recognition of alpha-galactosylceramide by human CD4(+) and CD8alphabeta(+) T lymphocytes

Human CD1d molecules present an unknown ligand, mimicked by the synthetic glycosphingolipid alpha-galactosylceramide (alphaGC), to a highly conserved NKT cell subset expressing an invariant TCR Valpha24-JalphaQ paired with Vbeta11 chain (Valpha24(+)Vbeta11(+) invariant NK T cell (NKT(inv))). The developmental pathway of Valpha24(+)Vbeta11(+)NKT(inv) is still unclear, but recent studies in mice were consistent with a TCR instructive, rather than a stochastic, model of differentiation. Using CD1d-alphaGC-tetramers, we demonstrate that in humans, TCR variable domains other than Valpha24 and Vbeta11 can mediate specific recognition of CD1d-alphaGC. In contrast to Valpha24(+)Vbeta11(+)NKT(inv) cells, Valpha24(-)/CD1d-alphaGC-specific T cells express either CD8alphabeta or CD4 molecules, but they are never CD4 CD8 double negative. We show that CD8alphabeta(+)Valpha24(-)/CD1d-alphaGC-specific T cells exhibit CD8-dependent specific cytotoxicity and have lower affinity TCRs than Valpha24(+)/CD1d-alphaGC-specific T cells. In conclusion, our results demonstrate that, contrary to the currently held view, recognition of CD1d-alphaGC complex in humans is not uniformly restricted to the Valpha24-JalphaQ/Vbeta11 NKT cell subset, but can be mediated by a diverse range of Valpha and Vbeta domains. The existence of a diverse repertoire of CD1d-alphaGC-specific T cells in humans strongly supports their Ag-driven selection.     

CTLA-4 and CD28 mRNA are coexpressed in most T cells after activation. Expression of CTLA-4 and CD28 mRNA does not correlate with the pattern of lymphokine production.

Ag-presenting cells provide at least two distinct signals for T cell activation. T cell receptor-dependent stimulation is provided by presentation of a specific peptide Ag in association with MHC molecules. In addition, APC also supply costimulatory signals required for T cell activation that are neither Ag- nor MHC restricted. One such costimulatory signal is mediated via the interaction of B7 on APC with the CD28 receptor on T cells. Recently, CTLA-4 has been shown to be a second B7 receptor on T cells. In the present report, we have examined the expression of CD28 and CTLA-4 on a panel of resting and activated normal T cell subsets and T cell clones by RNA blot analysis in an attempt to determine whether their expression defines reciprocal or overlapping subsets. CD28 was detected in resting T cells, whereas CTLA-4 was not. After stimulation with PHA and PMA for 24 h, CTLA-4 mRNA was expressed in both the CD4+ and CD8+ subsets as well as in CD28+ T cells. We examined 37 human and six murine T cell clones that had been previously characterized for their cytokine production. After activation, CTLA-4 and CD28 mRNA were coexpressed in 36 of 37 human T cell clones and all six murine T cell clones. These included T cells of CD4+8-, CD4-8+, and CD4-8- phenotypes as well as clones with Th1 and Th2 cytokine profiles. In contrast, CD28 but not CTLA-4 mRNA was detected in leukemic T cell lines and myelomas. CTLA-4 and B7 mRNA but not CD28 mRNA was detected in two long term HTLV-I-transformed T cell lines. These data demonstrate that CD28 and CTLA-4 mRNA are coexpressed in most activated T cells and T cell clones, providing evidence that they do not define reciprocal subsets. Moreover, they are consistent with the hypothesis that B7 transmits its signal through a single receptor, CD28, on resting T cells, and multiple receptors, CD28 and CTLA-4, on activated T cells.     

Herpes simplex virus glycoprotein D is recognized as antigen by CD4+ and CD8+ T lymphocytes from infected mice. Characterization of T cell clones

Several previous reports have described the surprising inability to detect murine CTL specific for glycoprotein D (gD), one of the important protective immunogens of HSV. Using slight variations of published procedures, we were able to show that the immune response to HSV in infected mice includes the generation of CTL specific for gD. C3H/OuJ (H-2k) mice were infected by injection in the hind footpads with purified HSV-1. Lymphocytes from draining lymph nodes were then isolated and shown to proliferate in response to, and to kill, transformed fibroblasts (H-2k) expressing HSV-1 gD. Two gD-specific T cell clones were isolated. One clone, designated CGD1, was shwon to be CD8+. This clone recognizes HSV-1 gD, but not HSV-2 gD, in the context of class I MHC molecules and kills the appropriate MHC-matched fibroblasts expressing HSV-1 gD. Unusual features of this cytolytic clone include augmentation by IL-4 of proliferative responses to Ag, inhibition of its lytic activity by a mAb specific for Thy-1 and recognition of infected fibroblasts in preference to infected lymphoblasts. The other clone, designated CGD3, was shown to be CD4+. This clone recognizes both HSV-1 gD and HSV-2 gD in the context of class II MHC molecules and has cytolytic potential.     

Effect of candidate vaginally-applied microbicide compounds on recognition of antigen by CD4+ and CD8+ T lymphocytes

Vaginally applied antimicrobial compounds (microbicides) are being developed as an alternative method for preventing the spread of sexually transmitted diseases. In addition to identifying compounds effective against a spectrum of sexually transmitted pathogens, it will be important to ensure that these compounds are safe. Avoiding toxicity, inflammatory responses, or alteration of the function of resident immune cells are important considerations for the development of vaginally applied microbicides. Studies were performed with two classes of candidate microbicide compounds to determine if they would interfere with the recognition of antigen by CD4(+) and CD8(+) T lymphocytes. The presence of nontoxic concentrations of the anionic detergent cholic acid or the sulfated polymer lambda carrageenan did not inhibit recognition of immune peptide by antigen-specific T cells. However, antigen recognition by both CD4(+) and CD8(+) T lymphocytes was inhibited in the presence of the naphthalene sulfonate polymer PRO 2000. Brief (4-h) exposure of antigen-presenting cells or T cells to PRO 2000 did not result in inhibition of antigen uptake and processing by antigen-presenting cells or the ability of specific T cells to respond to antigen stimulation, suggesting that the inhibition was temporary. Binding of antibodies specific for CD18, CD8, and CD3 was impaired in the presence of PRO 2000, suggesting that the mechanism by which this microbicide inhibits T cell recognition of antigenic peptide may involve masking or internalization of surface proteins involved in T cell signaling or stabilizing T cell-antigen-presenting cell interactions. The assays described in this study represent a useful means to screen candidate topical microbicide compounds for inappropriate interactions with immune cells and may be useful for prioritization of candidate microbicide compounds.     

Correlation between the expression of CD4 and the level of CD4 mRNA in human B-cell lines

Lines of Epstein-Barr virus (EBV)-transformed lymphoblastoid B-cells (B-LCLs) differ in the expression of surface CD4 glycoproteins. The aim of the present study was to correlate the expression of CD4 molecules on B-LCL cells with the synthesis of CD4 mRNA. RT-PCR assays were performed with oligonucleotide primers designed to detect mRNA corresponding to intracellular, transmembrane, or extracellular portions of the CD4 molecule. RT-PCR assays with all sets of primers were positive in T-cell populations, but were negative in various B-cell lymphoma lines. The majority of the LCLs established by EBV transfection of non-selected B-cells yielded positive results with at least some of the primer sets used for detection of CD4 mRNA. A significant positive correlation was found between the proportion of CD4+ cells in various B-LCLs and the concentration of CD4 mRNA. LCLs established from B-cells which synthesized various antibodies did not express CD4 molecules and either failed to synthesize CD4 mRNA or produced very low concentrations. These findings indicate that the expression of CD4 on B-LCLs is directly correlated with the concentration of CD4 mRNA synthesized and with the differentiation stage in which B-cells were immortalized by EBV infection.     

The CD4 and CD8 T cell surface antigens are associated with the internal membrane tyrosine-protein kinase p56lck

The CD4 and CD8 T cell antigens are thought to transduce an independent signal during the process of T cell activation. We report our evaluation of the possible involvement of the lymphocyte-specific tyrosine kinase p56lck in these transduction pathways. Our data demonstrate that p56lck is specifically modulated with either CD4 or CD8 following antibody-mediated cross-linking of these molecules and that a large fraction of the total cellular lck protein can be coimmunoprecipitated with these surface glycoproteins. These results suggest that p56lck is functionally and physically associated with CD4/CD8 in normal murine T lymphocytes and support the concept that an independent signal is transduced by the interaction of these surface molecules with major histocompatibility complex determinants.     

Selective retention of activated CD8+ T cells by the normal liver.

Activation-induced cell death resulting in peripheral deletion of CD8+ T cells is associated with the accumulation of large numbers of apoptotic T cells in the liver. The hypothesis that this accumulation results from the intrahepatic trapping of T cells from the circulating pool predicts that the liver should retain T cells, which subsequently undergo apoptosis. Here we test this prediction. Perfusion of the liver with lymphocyte mixtures showed retention of activated, but neither resting nor apoptosing, T cells. This trapping was selective for CD8+ cells and was mediated primarily by ICAM-1 constitutively expressed on sinusoidal endothelial cells and Kupffer cells. T cells trapped in the liver became apoptotic. The normal liver is therefore a "sink" for activated T cells.     

Differential regulation of CD43 glycoforms on CD4+ and CD8+ T lymphocytes in graft-versus-host disease

Two distinct T-cell glycoforms of CD43 result from differentialglycosylation of a single gene product in vivo. The 115 kDaglycoform carries mainly tetrasaccharides and is a pan T-cellmarker, whereas the 130 kDa glycoform carries mainly hexasaccharidesand is associated with T-cell activation. CD43 has been shownto play a role both in enhancing and inhibiting cell adhesion;however, the function of the individual glycoforms is unknown.We have examined the distribution and regulation of the CD43glycoforms in a murine model of acute graft-versus-host disease(GVHD) using monoclonal antibodies (mAbs) S7 and 1B11 specificfor the 115 and 130 kDa CD43 glycoforms, respectively. An increasein T-lymphocyte CD43 130 kDa expression occurred during GVHDfrom day 4 onwards and coincided with splenomegaly and upregulationof the ß1-6GlcNAc transferase (C2GnT), the key enzymeresponsible for the addition of complex O-glycan branching toCD43. When T-lymphocyte subsets were examined for CD43 expression,we found that in GVHD, both CD43 glycoforms were upregulatedon CD4⁺ T cells. However, in CD8⁺ T cells, CD43 115 kDa wasdownregulated while CD43 130 kDa was dramatically upregulated,such that two distinct CD8⁺1B11⁺ T-cell subsets were observed.These data demonstrate differential expression of the CD43 glycoformsin both resting and activated CD4⁺ and CD8⁺ T cells, and suggestthat glycosylation differences between the CD43 glycoforms mayreflect participation in the different functions of these T-cellsubsets in immune disorders in vivo. activation CD43 glycosyltransferases graft-versushost disease T lymphocytes